Cells must change the activity of their genes in response to environmental cues, such as occur when a bacterium infects a person or when neurons connect with each other in the developing brain. Many controlling RNAs function as small switches, that instruct the cell to respond to signals such as a change in pH or a chemical secreted by nearby cells. The operation of a "genetic switch" often involves a change in the 3D shape of one RNA or base pairing between two antisense RNAs. The main goals of this research are to understand (1) the physical forces that control the structures of RNA molecules and (2) how changes in RNA structure can switch gene expression on and off.The outcome of this research will improve human health, by providing basic knowledge of how bacteria adapt to the environment of their human host and enabling the rational design of artificial RNA sensors and switches for therapeutic applications. Two problems that will be addressed by this proposal are (1) how helix switching is coupled to RNA tertiary structure and (2) how proteins promote RNA strand exchange and the hybridization of natural antisense RNAs with their targets. The study of the folding pathway of the Tetrahymena ribozyme by ourselves and others has revealed general principles of RNA folding mechanisms. These principles and the experimental methods developed for the study of RNA folding reactions will be applied to the problem of conformational switching during RNA regulation. In aim 1, stopped-flow fluorescence spectroscopy will be used to investigate the coupling of tertiary and secondary structure during folding of the PSabc domain of the Tetrahymena intron. These studies will provide basic information about RNA folding transition states. Aim 2 will determine RNA secondary and tertiary interactions required for regulation of E. coli rpoS mRNA by the small regulatory RNA DsrA and the bacterial Sm-like protein Hfq. In aim 3, fluorescent 'molecular beacons'and small RNA substrates will be used to investigate how Hfq protein facilitates RNA annealing and strand exchange reactions. Hfq regulates mRNA stability in bactria and is necessary for the function of more than 20 natural antisense RNAs. The results of this study will be directly relevant to the function of human Sm and Lsm proteins, which are homologous to Hfq, and which are vital to many stages of pre-mRNA splicing and mRNA expression.